scholarly journals Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

2000 ◽  
Vol 66 (7) ◽  
pp. 2811-2816 ◽  
Author(s):  
Yasuhiro Mihara ◽  
Takashi Utagawa ◽  
Hideaki Yamada ◽  
Yasuhisa Asano
Keyword(s):  
Acid Phosphatase ◽  
Random Mutagenesis ◽  
Reaction Yield ◽  
Mutant Library ◽  
Morganella Morganii ◽  
Gene Encoding ◽  
E Coli ◽  
Acid Phosphatase Gene ◽  
Phosphate Source

ABSTRACT A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. TheMorganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to theM. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced intoEscherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreasedKm value for inosine was responsible for the increased productivity.

scholarly journals A Kayvirus Distant Homolog of Staphylococcal Virulence Determinants and VISA Biomarker Is a Phage Lytic Enzyme

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 292
Author(s):  
Aleksandra Głowacka-Rutkowska ◽  
Magdalena Ulatowska ◽  
Joanna Empel ◽  
Magdalena Kowalczyk ◽  
Jakub Boreczek ◽  
...  
Keyword(s):  
Cell Walls ◽  
Bacterial Transport ◽  
Lytic Enzyme ◽  
Virulence Determinants ◽  
Gene Encoding ◽  
Phage Gene ◽  
E Coli ◽  
Lytic Transglycosylase ◽  
Distant Homolog

Staphylococcal bacteriophages of the Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to two staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs IsaA and SceD. We show that Tgl is a lytic enzyme secreted by the bacterial transport system and localizes to cell peripheries like IsaA and SceD. It causes lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destroy S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from the lysK gene, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, like phage early genes. Taken together, our data indicate that tgl belongs to the kayvirus lytic module and encodes an additional endolysin that can act in concert with LysK in cell lysis.

scholarly journals Regulation and Mechanism of Action of the Small Heat Shock Protein from the Hyperthermophilic ArchaeonPyrococcus furiosus

2001 ◽  
Vol 183 (17) ◽  
pp. 5198-5202 ◽  
Author(s):  
Pongpan Laksanalamai ◽  
Dennis L. Maeder ◽  
Frank T. Robb
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mechanism Of Action ◽  
Pyrococcus Furiosus ◽  
Small Heat Shock Protein ◽  
Control Culture ◽  
Gene Encoding ◽  
E Coli

ABSTRACT The small heat shock protein (sHSP) from the hyperthermophilePyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105°C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105°C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56°C. Survivability of E. colioverexpressing the sHSP was enhanced approximately sixfold during exposure to 50°C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.

scholarly journals Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Structural Studies ◽  
Gene Encoding ◽  
E Coli ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

scholarly journals Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

2021 ◽  
Vol 11 ◽  
Author(s):  
Ashwini Kumar Ray ◽  
Paula B. Luis ◽  
Surabhi Kirti Mishra ◽  
Daniel P. Barry ◽  
Mohammad Asim ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Growth Inhibition ◽  
Mrna Expression ◽  
Host Protein ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
H Pylori

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

Effects of Escherichia coli endotoxin on rat platelets

1962 ◽  
Vol 203 (2) ◽  
pp. 296-298 ◽  
Author(s):  
Albert J. Roy ◽  
Isaac Djerassi ◽  
Harvey Neitlich ◽  
Sidney Farber
Keyword(s):  
Escherichia Coli ◽  
Platelet Count ◽  
Acid Phosphatase ◽  
Osmotic Fragility ◽  
Rat Plasma ◽  
E Coli ◽  
In Vitro Incubation ◽  
Rat Platelets

Administration of E. coli endotoxin to normal rats results in a gradual and profound decrease of the levels of circulating platelets. In vitro incubation of fresh platelet-rich rat plasma with this endotoxin does not affect the platelet count. The osmotic fragility of the platelets, determined by release of acid phosphatase is, however, increased. The survival of endotoxin-treated platelets, estimated by their ability to circulate in thrombocytopenic rats, is markedly reduced.

Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress

2010 ◽  
Vol 426 (3) ◽  
pp. 271-280 ◽  
Author(s):  
Arvind Anand ◽  
Brian T. Duk ◽  
Sandeep Singh ◽  
Meltem Y. Akbas ◽  
Dale A. Webster ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcriptional Control ◽  
High Sensitivity ◽  
Reduced Form ◽  
Gene Encoding ◽  
Antioxidant Genes ◽  
E Coli ◽  
Hypoxic Conditions ◽  
Catalase Peroxidase

The bacterial haemoglobin from Vitreoscilla, VHb, displays several unusual properties that are unique among the globin family. When the gene encoding VHb, vgb, is expressed from its natural promoter in either Vitreoscilla or Escherichia coli, the level of VHb increases more than 50-fold under hypoxic conditions and decreases significantly during oxidative stress, suggesting similar functioning of the vgb promoter in both organisms. In the present study we show that expression of VHb in E. coli induced the antioxidant genes katG (catalase–peroxidase G) and sodA (superoxide dismutase A) and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of the oxidative stress regulator OxyR in mediating the protective effect of VHb under oxidative stress. A putative OxyR-binding site was identified within the vgb promoter and a gel-shift assay confirmed its interaction with oxidized OxyR, an interaction which was disrupted by the reduced form of the transcriptional activator Fnr (fumurate and nitrate reductase). This suggested that the redox state of OxyR and Fnr modulates their interaction with the vgb promoter. VHb associated with reduced OxyR in two-hybrid screen experiments and in vitro, converting it into an oxidized state in the presence of NADH, a condition where VHb is known to generate H2O2. These observations unveil a novel mechanism by which VHb may transmit signals to OxyR to autoregulate its own biosynthesis, simultaneously activating oxidative stress functions. The activation of OxyR via VHb, reported in the present paper for the first time, suggests the involvement of VHb in transcriptional control of many other genes as well.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

scholarly journals A nadA Mutation Confers Nicotinic Acid Auxotrophy in Pro-carcinogenic Intestinal Escherichia coli NC101

2021 ◽  
Vol 12 ◽  
Author(s):  
Lacey R. Lopez ◽  
Cassandra J. Barlogio ◽  
Christopher A. Broberg ◽  
Jeremy Wang ◽  
Janelle C. Arthur
Keyword(s):  
Escherichia Coli ◽  
Nicotinic Acid ◽  
De Novo ◽  
Host Cells ◽  
Gene Encoding ◽  
E Coli ◽  
Functional Studies ◽  
Bowel Diseases ◽  
Mechanistic Investigations

Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.

scholarly journals Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

1998 ◽  
Vol 18 (6) ◽  
pp. 3289-3299 ◽  
Author(s):  
Dongqing Huang ◽  
Jason Moffat ◽  
Wayne A. Wilson ◽  
Lynda Moore ◽  
Christine Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acid Phosphatase ◽  
Protein Kinase ◽  
Substrate Specificity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
Acid Phosphatase Gene

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

Effect of Copper on Acid Phosphatase Activity in Yeast Yarrowia lipolytica

2007 ◽  
Vol 62 (1-2) ◽  
pp. 70-76 ◽  
Author(s):  
Hiroyasu Ito ◽  
Masahiro Inouhe ◽  
Hiroshi Tohoyama ◽  
Masanori Joho
Keyword(s):  
Acid Phosphatase ◽  
Yarrowia Lipolytica ◽  
Acid Phosphatase Activity ◽  
Inorganic Phosphate ◽  
Soluble Form ◽  
Gene Encoding ◽  
Effect Of Copper ◽  
Yeast Yarrowia Lipolytica ◽  
Apase Activity

Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu2+ concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu2+, Co2+, Ni2+, Mn2+ and Mg2+ and inhibited by Ag+ and Cd2+. The most effective ion was Cu2+, especially for the enzyme from cultures in medium containing Cu2+, whereas APase activity in wall-bound fragments was only slightly activated by Cu2+. The content of cellular phosphate involving polyphosphate was decreased by adding Cu2+, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu2+ might depend on derepression of the gene encoding the APase isozyme.

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